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Abstract

Background

Chorioamniotic membranes infection is a pathologic condition in which an abnormal
secretion of proinflammatory cytokines halts fetal immune tolerance. The aim of the
present study was to evaluate the functional response of human chorioamniotic membranes,
as well as the individual contribution of the amnion and choriodecidua after stimulation
with Escherichia coli, a pathogen associated with preterm labor.

Methods

Explants of chorioamniotic membranes from 10 women (37–40 weeks of gestation) were
mounted and cultured in a Transwell system, which allowed us to test the amnion and
choriodecidua compartments independently. Escherichia coli (1 × 10 6 CFU/mL) was added
to either the amniotic or the choriodecidual regions or both; after a 24-h incubation,
the secretion of IL-1beta, IL-6, TNFalpha, IL-8, and IL-10 in both compartments was
measured using a specific ELISA. Data were analyzed by Kruskal-Wallis one-way analysis
of variance.

Results

After stimulation with Escherichia coli, the choriodecidua compartment showed an increase
in the secretion of IL-1beta (21-fold), IL-6 (2-fold), IL-8 (6-fold), and IL-10 (37-fold),
regardless of which side of the membrane was stimulated; TNFalpha secretion augmented
(22-fold) also but only when the stimulus was applied simultaneously to both sides.
When the amnion was stimulated directly, the level of IL-1beta (13-fold) rose significantly;
however, the increase in IL-8 secretion was larger (20-fold), regardless of the primary
site of infection. TNFalpha secretion in the amnion compartment rose markedly only
when Escherichia coli was simultaneously applied to both sides.

Conclusion

Selective stimulation of fetal membranes with Escherichia coli results in a differential
production of IL-1beta, IL-6, TNFalpha, IL-8, and IL-10. These tissues were less responsive
when the amnion side was stimulated. This is in agreement with the hypothesis that
the choriodecidua may play a primary role during an ascending intrauterine infection,
being the main barrier to progression of the infection into the amniotic cavity. Therefore,
the tissue-specific capacities for the secretion of these immune modulators could
be a determining factor for the degree of severity of the inflammation process in
fetal membranes.

Background

Pregnancy is the result of a fine immunological privilege that allows the fetus to
co-habit the maternal uterus, preventing rejection of the fetal allograft [1]. There are epidemiologic and experimental data indicating that intrauterine infection
is a pathological condition that disrupts this privilege [2,3] and is the main etiological factor for the development of Premature Rupture of Membranes
(PROM). This pathology complicates about 30–40% of all preterm births [4] and 10% of all pregnancies [5].

Human chorioamniotic membranes form a complex multi-laminated tissue constituted by
the amnion whose epithelium is in contact with the amniotic fluid and the choriodecidua
that is formed by trophoblasts inter-digitized with the maternal decidua [5,6]. During an ascending infection, originated in the cervico-vaginal region [7-9], the choriodecidua is the first-line barrier in contact with the pathogens that can
cross the membranes and infect the amnion and the amniotic fluid [10]. In response, there is an abnormal production of proinflammatory cytokines, such
as interleukin (IL)-1β, tumor necrosis factor alpha (TNFα), IL-6, and IL-8 in the
extra-embryonic tissues, i.e., placenta [9] and chorioamniotic membranes [11-14].

In a previous work, using an ex-vivo model, we demonstrated that in vitro stimulation with lipopolysaccharide (LPS) and Streptococcus agalactiae induced a differential response in IL-1β and TNFα secretion by the amnion and choriodecidual
tissues [13]. Using the same model, the present study was aimed at: 1) Investigating the secretory
profiles of IL-1β, IL-6, TNFα, IL-8, and IL-10 by human chorioamniotic membranes after
stimulation with Escherichia coli, a common pathogen in cervical-vaginal infection in humans associated with pregnancy
losses [8,15], fetal cardiac dysfunction [16], neurological injury in preterm infants [17], as well as with preterm delivery and PROM [18]. 2) Disclosing whether or not a specific differential contribution by each membrane
exists.

Methods

The 10 pregnant women (37–40 weeks of gestation) studied were from an urban area of
Mexico City, 22–35 years old, previously normotensive, without history of diabetes
mellitus, thyroid, liver or chronic renal disease, cared for at the Obstetrics Outpatient
Service of the Instituto Nacional de Perinatologia. All women had uneventful pregnancies,
without evidence of active labor and with neither clinical nor microbiological signs
of chorioamnionitis or of lower genital tract infection. Samples were obtained after
delivery by elective cesarean section.

All women provided written, informed consent, before collection of samples. The institutional
Review Board approved the protocol and the collection and use of the samples and the
study was conducted according to the Guidelines on the Practice of Ethical Committees in Medical Research (3rd ed) issued by the Royal College of Physicians of London.

Fetal membrane explants culture

The chorioamniotic membranes were cut at a distance of 5–6 cm from the placental disc,
transported to the laboratory in sterile Dulbecco Modified Eagle Medium (DMEM; Gibco
BRL, Bethesda, MD), and rinsed in sterile saline solution to remove adherent blood
clots. Segments representing all zones of membranes were manually cut into 18 mm diameter
discs and held together with silicone rubber rings to be placed on the upper chamber
of a Transwell® system (CORNING, New York, NY) from which the original polycarbonate membrane had
been previously removed. In this model the upper chamber of the Transwell® system is delimited by choriodecidual tissue and the lower chamber by amniotic tissue,
which allowed testing the two compartments independently (Figure 1). A detailed description and validation of this model has been published previously
[13]. One milliliter of DMEM (Gibco BRL), supplemented with 10% fetal calf serum (FCS),
1 mM sodium pyruvate, and 1× antibiotic-antimycotic solution (penicillin 100 U/mL,
streptomycin 100 μg/mL) (DMEM-FCS) was added to each compartment. The mounted explants
were placed in a 12-well tissue culture plate (CORNING, New York, NY) and incubated
in 5% CO2 at 37°C for 24 h.

Figure 1.Experimental model. The fetal membranes are held together with silicone rubber rings in the upper chamber
of a Transwell® device. In this system, the choriodecidua region delimits the upper chamber and the
amnion region, the lower chamber.

Explants stimulation

To stabilize the explants after manipulation, they were pre-incubated for 24 h in
the medium (DMEM-FCS). Subsequently, the explants were washed with saline solution
to remove FCS and the remainder of the medium was changed to DMEM with 0.2% lactalbumin
hydrolysate (Gibco BRL) and co-incubated with 1 × 106 colony forming units (CFU) of Escherichia coli isolated from cervicovaginal exudates.

Four Each experiment (n = 10) included the following set of chambers, in triplicate:

Basal (n = 10),

control membranes in which only the medium culture was added to the compartments;

Choriodecidua (n = 10),

Escherichia coli was added only to the choriodecidua side;

Amnion

(n = 10),

E. coli was added only to the amniotic compartment;

Both

(n = 10),

the bacterium was added simultaneously to both compartments. After 24 h of co-incubation,
the medium from the amnion and choriodecidua chambers was collected and centrifuged
at 5,000 rpm, 3 min at 4°C, to precipitate Escherichia coli, and the bacterium-free medium of each sample was stored at -70°C until assayed.
Protein concentration in all samples was measured according to the Bradford method
[19].

Cytokine assays

IL-1β, TNF-α, IL-6, and IL-10 concentrations were quantified using specific DuoSet® enzyme-linked immunosorbent sandwich assays (ELISA) (R & D Systems, Minneapolis, USA).
For the IL-1β assay, a standard curve was developed from 4 to 260 pg/mL and the sensitivity
was 2 pg/mL; the TNF-α assay was linear from 15 to 960 pg/mL and sensitivity was 5.0
pg/mL; for the IL-6 assay, the curve was linear from 250 to 8000 pg/mL with a sensitivity
of 200 pg/mL; and for the IL-10 assay, a standard curve was developed from 1.25 to
2000 pg/mL, with a sensitivity of 1.0 pg/mL. IL-8 was measured using a commercial
kit (Amersham Biosciences, Buckinghamshire, UK) according to manufacturer's instructions.
The standard curve was linear from 25.6 to 1000 pg/mL and sensitivity was 5 pg/mL.

The final concentration of each cytokine was expressed per microgram of the total
protein concentration of each sample. Intra- and inter-assay coefficients of variation
were less than 5%. A rigorous quality control program, including external and internal
standards for all cytokines, is followed in our laboratory.

Statistical analysis

Since the results did not have a normal distribution, comparisons between the experimental
groups and the control were performed using the Kruskal-Wallis one-way analysis of
variance on rank tests. A P < 0.05 was considered significant. The data expressed in the text and figures represent
the median and the 95% confidence interval limits (95% CI).

Results

Basal secretion of IL-1β was similar in both the amniotic and the choriodecidua compartments
(0.8 [0.30–2.89] and 1.73 [0.12–3.27] pg/μg protein, respectively); it increased markedly
in the choriodecidua tissue (P < 0.05) regardless of whether Escherichia coli was applied directly to either the choriodecidua compartment (39.95 [26.9–55.6] pg/μg
protein) or the amniotic compartment (28.9 [23.0–50.5] pg/μg protein), or simultaneously
to both compartments (44.87 [21.87–50.5] pg/μg protein). However, IL-1β secreted by
the amnion rose slightly only when the bacterium was directly applied to this compartment
(13.9 [11.2–18.3] pg/μg protein) (Figure 2).

Figure 2.In vitro secretion of IL-1β in amnion and choriodecidua regions after selective stimulation
with Escherichia coli. IL-1β secreted to the culture medium after 24 h of infection with 1 × 106 CFU of E. coli. Data were normalized in function of protein concentration (pg/μg protein) and each
bar represents the 95% confidence intervals and the median (solid line) of 10 different
experiments. Significant difference between basal and stimulated values is indicated
(*P < 0.05) C. Choriodecidua; A. Amnion.

IL-6 (Figure 3) and IL-10 (Figure 4) showed a similar secretion profile, with the choriodecidua as the most active tissue
after Escherichia coli stimulation whether applied directly or indirectly to this membrane (P < 0.05). In
both cases, the largest response was observed when the stimulus was applied directly
in the choriodecidual zone (13.45 [9.39–18.3] and 32.38 [23.6–44.2] pg/μg protein,
respectively). Again, the amnion showed only a mild but significant increase in the
secretion of IL-6 and IL-10 (10.8 [8.3–16.75] and 15.97 [13.02–21.49] pg/μg protein,
respectively).

Figure 3.In vitro secretion of IL-6 in amnion and choriodecidua regions after selective infection
with Escherichia coli. IL-6 secreted to the culture medium after 24 h of infection with 1 × 106CFU of E. coli. Data were normalized in function of protein concentration (pg/μg protein) and each
bar represents the 95% confidence intervals and the median (solid line) of 10 different
experiments. Significant difference between basal and stimulated values is indicated
(*P < 0.05) C. Choriodecidua; A. Amnion.

Figure 4.In vitro secretion of IL-10 in amnion and choriodecidua regions after selective infection
with Escherichia coli. IL-10 secreted to the culture medium after 24 h of infection with 1 × 106CFU of E. coli. Data were normalized in function of protein concentration (pg/μg protein) and each
bar represents the 95% confidence intervals and the median (solid line) of 10 different
experiments. Significant difference between basal and stimulated values is indicated
(*P < 0.05) C. Choriodecidua; A. Amnion.

The basal secretion of TNFα by the amnion and choriodecidua was similar (Figure 5). However, the secretion by the choriodecidua tissue showed a 22-fold increase (53.15
[40.0–94.2] pg/μg protein) only when the Escherichia coli stimulus was applied simultaneously to both membranes (P < 0.05), while the amnion
showed a 25-fold increase (P < 0.05) (29.2 [14.5–35.3] pg/μg protein) (Figure 5).

Figure 5.In vitro secretion of TNF-α in amnion and choriodecidua regions after selective infection
with Escherichia coli. TNF-α secreted to the culture medium after 24 h of infection with 1 × 106 CFU of E. coli. Data were normalized in function of protein concentration (pg/μg protein) and each
bar represents the 95% confidence intervals and the median (solid line) of 10 different
experiments. Significant difference between basal and stimulated values is indicated
(*P < 0.05) C. Choriodecidua; A. Amnion.

The secretion profile of IL-8 was different from that of the other cytokines. Regardless
of the primary side of stimulation with Escherichia coli, the secretion was markedly increased in both compartments; however, the amnion was
the most active region with a 19-fold increase (P < 0.05) in comparison with its basal
concentration (Figure 6).

Figure 6.In vitro secretion of IL-8 in amnion and choriodecidua regions after selective infection
with Escherichia coli. IL-8 secreted to the culture medium after 24 h of infection with 1 × 106CFU of E. coli. Data were normalized in function of protein concentration (pg/μg protein) and each
bar represents the 95% confidence intervals and the median (solid line) of 10 different
experiments. Significant difference between basal and stimulated values is indicated
(*P < 0.05) C. Choriodecidua; A. Amnion.

Discussion

In the present study we used a previously published ex-vivo model that resembles the presence of two independent compartments with a fully functional
chorioamnion; its upper chamber is delimited by the choriodecidua and the lower chamber
by the amnion, allowing us to test both compartments simultaneously [13]. We included three different conditions of membrane stimulation with Escherichia coli, resembling clinical scenarios. 1) Microorganisms arrive to the choriodecidua through
ascendant colonization (choriodecidua stimulation), 2) bacteria reach the amnion side
by iatrogenic introduction during amniocentesis (amnion stimulation), and 3) bacteria
are in contact with both sides of the membranes at late stages of colonization, which
enables them to cross the membranes.

With this system, we were able to show that the basal secretions of IL-1β, IL-6, IL-10,
and IL-8 by the choriodecidual tissue increased significantly (P < 0.05) in response
to Escherichia coli stimulation, regardless of whether the stimulus was applied directly to this region,
the amnion, or to both regions simultaneously. In addition, TNFα increased markedly
in both the choriodecidua and the amnion sides when the bacterium was applied to both
membranes at the same time. IL-1β rose mildly when the amnion was directly stimulated
by Escherichia coli whereas IL-8 secretion rose markedly in the amnion regardless of the primary side
stimulated. A marked rise in TNFα secretion in the amnion side was also observed but
only when the bacterium was simultaneously applied to the amnion and choriodecidua
tissues.

Previous works have evidenced that inoculation of the amniotic cavity with Escherichia coli induces a toxic response, characterized by the overproduction of pro-inflammatory
cytokines [20,21], such as IL-1β, a potent immunomodulator able to induce preterm labor after its experimental
infusion in pregnant rhesus monkeys [22]. However, the used animal models do not allow characterizing the particular and specific
contribution of each of the chorioamniotic membranes in this response and cannot be
compared with our results. In addition, we demonstrated that in vitro stimulation of chorioamniotic membranes with lipopolysaccharide (LPS) and Streptococcus agalactiae induces IL-1β increase in the choriodecidua region [13].

On the other hand, the IL-6 concentration in the amniotic fluid is considered a marker
of intra-amniotic inflammation and is frequently associated with an infectious process
in either the amniotic fluid or the chorioamniotic space [23,24]. Previous evidence has demonstrated that the concentration of this cytokine is increased
in human decidua [25] and chorion cells [26] after treatment with IL-1β and TNFα.

Similar findings have been obtained with IL-10, an anti-inflammatory cytokine, to
which a role as therapeutic factor in preterm labor has been ascribed [27] and whose concentration increases in patients with preterm labor associated with
intrauterine infection [28].

The differential secretion capacities of pro-inflammatory cytokines by the amnion
and the choriodecidua after Escherichia coli stimulation here reported suggest the existence of a complex interactive regulation
network. There is evidence indicating that IL-1β [29], TNFα [30], IL-6 [31], and IL-10 [32], in different gestational tissues, are able to induce biosynthesis and secretion
of prostaglandin E2 (PGE2) and PGF2α; these uterotonic factors play key roles in the onset and progression of labor [33] in normal and pathological conditions. In a previous work, we demonstrated that the
amnion's epithelium is the main source of PGE2 secretion after stimulation with Candida albicans, yeast that has been associated with cervico-vaginal infections [14].

Under the experimental conditions used in our study, it was clear that IL-1β, IL-6,
and IL-10 were mainly secreted to the choriodecidual compartment regardless of the
primary zone stimulated with Escherichia coli. Interestingly, when the amnion was primarily stimulated, its contribution to increase
the secretion of theses cytokines was small if any; whereas the secretion of theses
cytokines by the choriodecidual region was significantly increased. This observation
suggests the existence of a communication or "cross-talk" between both regions and,
thus, it is possible that the amnion's epithelium might be an important indirect factor
in the whole cytokine response.

On the other hand, TNFα, whose negative effects on the course of pregnancy have been
characterized [34,35], increases in the amniotic fluid of women with preterm labor and intra-amniotic infection
[30]. There is also experimental evidence demonstrating that the administration of a TNFα
bolus to pregnant animals causes profuse hemorrhage and pregnancy termination [36,37]. In our model, the increase in TNFα secretion in both the amnion and choriodecidua
compartments was significant (P < 0.05) only after simultaneous stimulation of both
membrane sides. A possible interpretation of the biological significance of these
findings is that, since TNFα is a pro-inflammatory cytokine with major immuno-toxic
properties, the chorioamniotic membrane coordinates its secretion in response to a
very complicated infectious scenario, as represented by chorioamnionitis, in which
both the fetal and maternal sides are insulted by an infectious agent.

It is possible that the secretion of IL-1β, IL-6, IL-10, and TNFα in the choriodecidua
region after infection with Escherichia coli would favor their trans-membranal translocation to the amnion [38] and thereby exert their effect on the whole membrane.

The IL-8 secretion pattern was also interesting, because both the amnion and the choriodecidua
were active in the secretion of this chemokine. This ubiquitous production might play
a key role in the recruitment and activation of professional cells of the immune system,
such as neutrophils whose migration toward the cervix, the placenta, and chorioamniotic
membranes is a clinical/histological characteristic of infection/inflammation [39,40]. The present results show that the IL-8 response in the amnion was mild as compared
to the choriodecidua region, which is the first tissue to be colonized by the microbial
pathogen during an ascending intrauterine infection and is the main barrier to progression
of infection into the amniotic cavity. Therefore, the tissue-specific capacities could
be important factors in determining the severity of the inflammation in fetal membranes
infected with Escherichia coli.

It is tempting to hypothesize that Escherichia coli infection of the chorioamniotic membrane may induce a precocious onset of the overproduction
of pro-inflammatory cytokines (an "anticipation" of the normal parturition cascade?),
leading to PROM and preterm labor.

The choriodecidua is the most responsive region to the infection, as it is the first
tissue to be colonized by the microbial pathogen during an ascending intrauterine
infection and it is the main barrier to progression of the infection into the amniotic
cavity. Therefore, the tissue-specific capacities of this region to secrete different
proinflammatory cytokines are crucial factors for determining the severity of the
inflammation process of fetal membranes.

Authors' contributions

AEN, AFP, DSB and RMC carried out samples collection, ELISA assays and microbiologic
control. GGL carried out culture membranes and stimulation with bacterium. HFH coordinated
data collection and provided statistical analysis. VZC, FVO and HML participate in
the design of the study, data analysis and manuscript preparation. All authors read
and approved the final manuscript.

Acknowledgements

This work was supported by the National Council of Science and Technology of Mexico
(CONACyT, Grant No. 61590). We thank Dr. Adalberto Parra for his critical suggestions
to the manuscript.